Study of Oxidant (MDA) and SOD & Vitamin E in Hypertensive Patients & Normotensive Individuals by SSR-IIJLS JOURNAL

Int. J. Life. Sci. Scienti. Res., VOLUME 2, ISSUE 1, pp: 9-14

(ISSN: 2455-1716)

JANUARY-2016

Research Article (Open access)

Study of Oxidant (MDA) and Antioxidants (SOD & Vitamin E) in Hypertensive Patients and Normotensive Individuals Shashi Prabha Singh1*, Manish Kumar Verma1, Pratibha Tripathi1, Durgesh Singh2 1

Department of Biochemistry, Integral Institute of Medical Sciences & Research Lucknow, India 2

Department of Anatomy, Integral Institute of Medical Sciences & Research Lucknow, India

ABSTRACT- This study was undertaken to evaluate the serum levels of Oxidant (MDA) & antioxidant (SOD & Vitamin E) and compare oxidative stress (MDA) level among normotensive and hypertensive subjects. Oxidative stress has been relationship with mechanisms of EH (essential hypertension). A total number of 70 subjects were taken including both sex (Men and Women) between the ages of 35-70 years taken in this study. Exclusion criteria were chronic diseases, alcohol consumer, obesity, smoking/tobacco consumer and current use of any medication. Antioxidant enzymes activity and lipid peroxidation (malondialdehyde) were determined in serum. In 70 subjects out of 35 were found as an controls normotensive individuals and the cases 35 hypertensive patients. Serum MDA levels were highly significantly elevated in hypertensive patients in compared to normotensive individuals (4.39±0.98 µmol/l vs 1.51±0.70µmol/l and p < 0.0001). SOD acts as an antioxidant was highly significantly decrease in hypertensive patients in compared to normotensive individuals (0.44±0.06U/mg protein/min vs 0.96±0.04 U/mg protein/min and p <0.0001). Vitamin E, which acts as a biomarker of hypertensive was significantly higher decrease in hypertensive in compared to normotensive individuals (0.69±0.08 vs 1.06±0.25 and p < 0.001). These findings demonstrate the strong association of SOD and Vitamin E level decrease in hypertensive patients and by MDA level increase in hypertensive patients. Oxidative stress in hypertensive patients increasing over time may play a role in the improvement of atherosclerosis and cardiovascular disease, should be considered in further research.

Key Words: Hypertensive, Normotensive individuals, MDA, SOD, Vitamin E -------------------------------------------------IJLSSR-----------------------------------------------

INTRODUCTION Hypertension (HT) is a major health problem worldwide.

Excess of sugar and salt or deficiencies of antioxidant vitamins in

Individuals with hypertension are at an increased risk for stroke,

diet play a vital role in the etiology of hypertension.

heart disease, and kidney failure. Although the etiology of

Cardiovascular disease counted 2.3 million deaths in India in year

essential hypertension has a genetic component, lifestyle factors

1990; this is predicted to double by the year 2020. Hypertension

such as diet play an important role.

is directly responsible for 57% of all stroke death and 24% of all coronary heart disease in India. [1] Studies demonstrate that hypertension may develop as a result of

Corresponding Address

increased reactive oxygen species *Dr. Shashi Prabha Singh Department of Biochemistry Integral Institute of Medical Sciences & Research Lucknow, India E-mail: dr.spsingh084@gmail.com Received: 02 November 2015/Revised: 25 November 2015/Accepted: 26 December 2015

[2, 3]

and that a variety of

antioxidant therapies ameliorate hypertension. Hypertensive effects of oxidative stress are mostly due to endothelial dysfunction resulting from disturbances of vasodilator systems, particularly degradation of nitric oxide (NO) by oxygen-free radicals. [4-5] Reduced antioxidant capacity also promotes cellular oxidative

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Int. J. Life. Sci. Scienti. Res., VOLUME 2, ISSUE 1 stress and is implicated in cardiovascular and renal oxidative

METHODOLOGY

damage in hypertension. Superoxide dismutase (SOD) activities

The criteria used for selection of both hypertensive and

are reduced in hypertensive patients. An antioxidant SOD

normotensive controls were performed by well-established

catalyses the conversion of superoxide radicals to H2O2 and O2

diagnostic criteria as recommended by 7th Joint National

protecting the cells against potential toxicity of reactive oxygen.

Committee. The present study was conducted on cases of 35

H2O2 is further detoxified by catalase.

[6]

hypertensive patients and was control 35 normotensive. The

Hypertension, on the other hand, may lead to tissue damage

study was approved by the Institute Ethics Committee, Integral

through lipid per oxidation and other oxidative mechanisms. In

Institute of Medical Sciences & Research Lucknow, India and

vivo oxidation of low-density lipoproteins by oxygen-free

informed consent was obtained from all the cases and control

radicals may increase hypertension-related atherogenesis, and

subjects. A venous blood sample was collected by using

antioxidants may be beneficial in this regard. Studies concerning

disposable syringes. A volume of 3ml of blood is collected by

associations between serum levels of antioxidants and hyperten-

venipuncture under aseptetic conditions in a sterile clot activator/

sion have been inconsistent.

[7]

plain vial from selected subjects by the

investigator. Blood in

As recommended by the American Institute of Nutrition, the daily

the tube was allowed to clot at room

temperature for 10-15

amount of vitamin E in humans is 30 IU/d or 0.43 IU/kg per day.

minutes and then centrifuged at (4000 rpm) for approx 2-3

Current clinical criteria for defining hypertension generally are

minutes. After centrifugation, supernatant (serum) was collected

based on the average of two or more seated blood pressure

and split into 3 micro tubes for the study of MDA, SOD and

readings during each of two or more outpatient visits.[8] Based on

vitamin E.

the seventh report of the Joint National Committee on prevention,

The serum samples were used for the analysis of various

detection, evaluation and treatment of high blood pressure

parameters:

(JNC VII report).

Estimation of Malondialdehyde by Satoh K. (1978)

Blood Pressure is classified into the following stages: category

Systolic blood pressure(SBP) mm of Hg

method [10]

Diastolic blood

The TCA –TBA-HCl solution was freshly prepared by mixing

pressure(SBP) mm

equal volume of 15% TCA, 0.375% TBA and 0.25N HCl. 0.8 ml

of Hg

of serum +1.2 of TCA-TBA-HCl reagent. Mixed immediately + kept in a boiling water bath for 10 minutes. Cooled +2ml of 1N

Normal

<120

<80

Prehypertension

120-139

80-89

Hypertension, stage I

140-159

90-99

Hypertension, stage II

≥ 160

≥100

NaOH (freshly prepared) to eliminate centrifugation. O.D at 535nm against blank which contained normal saline in place of serum The MDA concentration was calculated according to the following formula: MDA (µmol/l) =OD 532 × 1.75/0.15

Production of reactive oxygen species is a particularly destructive

Estimation of Superoxide dismutase (SOD) using

aspect of oxidative stress. Such species include free radicals and

Nitroblue tetrazolium (NBT) method [11]

peroxides. Some of the less reactive of these species (such as superoxide) can be converted by oxido-reduction reactions with transition metals or other redox cycling compounds (including quinones) into more aggressive radical species that can cause extensive cellular damage.[9]

The assay mixture contained 1.2ml of sodium pyrophosphate buffer, 0.1ml of PMS, 0.3ml of NBT, 0.2ml of the enzyme preparation and water in a total volume of 2.8ml. The reaction will be initiated by the addition of 0.2ml of NADH. The mixture will be incubated at 30oC for 90 seconds and arrested by the addition of 1.0ml of glacial acetic acid. The

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Int. J. Life. Sci. Scienti. Res., VOLUME 2, ISSUE 1 reaction

mixture will be then

shaken

with 4.0ml of

n-butanol, allowed to stand for 10 minutes and centrifuged. The intensity of the chromogen in the butanol layer was

Table-1: Comparison of MDA level between cases and controls Study variable

measured at 560nm in a spectrophotometer. One unit of enzyme activity is defined as the amount of enzyme that

MDA (µmol/L)

Controls

Cases

(N=35)

1.51±0.70

4.39±0.98

t- value

p- value

14.1476

<0.0001

gave 50% inhibition of NBT reduction in one minute. One unit of SOD activity is the amount of the enzyme

Table 1 shown the comparison of MDA level between cases and

that inhibits the rate of auto oxidation of NBT by 50% and

controls. MDA was significantly (p=0.0001) higher among cases

was expressed as Units /mg protein/min. The enzyme unit can

(4.39±0.98) than controls (1.51±0.70).

be calculated by using the following equation :

Table 2: Comparison of SOD level between cases and

Rate (R) = (final OD - initial OD) / 3 min, % of inhibition = {(blank OD - R) / blank OD} x 100

controls Study variable

Enzyme unit (U) = (% of inhibition / 50) x common

Controls

Cases (N=35)

t- value

p- value

0.44±0.06

42.6615

<0.0001

(N=35)

dilution factor

SOD(U/mg

[50% inhibition = 1 U]

protein/min)

0.96±0.04

Specific activity = (U / mg) protein

Estimation of Vit E by Emmoria engel reaction method

[12]

Table-2 shows the comparison of SOD level between cases and controls. SOD was significantly (p=0.0001) lower among cases

1 ml of plasma is thoroughly mixed with 1 ml of redistilled 95%

(0.44±0.06) as compared with controls (0.96±0.04).

ethanol in a 15 ml centrifuge tube (stoppered). 3 ml of petroleum

Table 3: Comparison of Vitamin E level between cases

ether is added to the tube and shaken vigorously for 3 minutes

and controls

and stoppered. 2 ml of clear supernatant in a clean dry cuvette is

Study

Controls

Cases

taken and the O.D is measured at 450 nm for carotenes. The

variable

(N=35)

petroleum ether is evaporated at low temperature (50 C) and low

Vitamin E

1.06±0.25

0.69±0.08

pressure. The residue is redissolved in 1 ml of chloroform. 1 ml

(mg/dl)

of 95% ethanol is added followed by 1 ml of α,α- dipyridyl followed by 0.1 ml of 0.1% FeCl3 exactly after 15 minutes. O.D is read at 520 nm and the concentration is calculated using the formula. OD at 520 nm – (OD at 450 nm x 0.29) X amount of standard X 100 OD of standard at 520 nm

volume of test

p- value

8.3392

<0.0001

Table-3 shows the comparison of vitamin E level between cases and controls. Vitamin E level was significantly (p=0.0001) lower among cases (0.69±0.08) as compared with controls (1.06±0.25).

Table-4: Pearson correlation coefficient among the study parameters in cases

Statistical analysis

MDA

The results are presented in mean ±SD and percentage. Unpaired

t-value

MDA

Pearson Correlation

SOD Vitamin E .42*

-.039

.010

.823

t-test was used to compare the study parameters between cases

Sig. (2-tailed)

and controls. Pearson correlation coefficient was calculated

Pearson Correlation

.428*

.004

Sig. (2-tailed)

.010

Pearson Correlation

-.039

.004

among the study parameters. P-value<0.05 was considered

SOD

significant.

.984

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Int. J. Life. Sci. Scienti. Res., VOLUME 2, ISSUE 1 Sig. (2-tailed)

.823

.984

*. Correlation is significant at the 0.05 level (2-tailed).

Only SOD and MDA was moderately correlated (r=0.42, p=0.01) in cases (Table 4 & Fig. 4).

Fig.5: Scatter diagram showing association between SOD and MDA in controls

DISCUSSION In the present study, MDA was significantly (p<0.0001) higher among cases (4.39±0.98) than controls (1.51±0.70) and this was in accordance with that of Fig. 4: Scatter diagram showing association between SOD

[14]

[13]

Similar finding was also reported

in which MDA was significantly higher in hypertensive

and MDA in cases

patients as compared to controls (p<0.05). Essential hypertension

Table 5: Pearson correlation coefficient among the

is associated with increased production of ROS predisposing to

study parameters in controls

increase in lipid peroxidation which is a marker for cellular

MDA

Pearson Correlation

MDA

SOD

Vitamin E

damage. An imbalance in the challenge posed by the increased

-.52**

.104

production of free radical mainly superoxide ions or decreased

.001

.565

Sig. (2-tailed)

SOD

Vitamin E

production of nitric oxide may facilitate the development of functional arterial spasm.

[15]

MDA can exacerbate the actions of

Pearson Correlation

-.52**

.124

Sig. (2-tailed)

.001

resulting in decreased dismutation of hydrogen peroxide, and

Pearson Correlation

.104

.124

consequently, increase in H2O2 concentration inactivates SOD

Sig. (2-tailed)

.565

.492

superoxide ions by impairing endothelium dependent relaxation and propagation of lipid peroxidation by chain reaction in membranes. Superoxide ions can inactivate calatalse enzyme,

leading to further rise in MDA levels. 33

**. Correlation is significant at the 0.01 level (2-tailed).

[16]

The increase in MDA

levels further inactivates the antioxidant enzymes (SOD) in untreated hypertension

[16, 17]

reported the increased damage of

various proteins in essential hypertension. The consequences of Only SOD and MDA was as correlation (r=-0.52, p=0.001) in

such oxidative protein damage in hypertension may also be one

controls (Table 5 & Fig. 5).

of the causes for reduced enzymatic activity. SOD was significantly (p<0.0001) lower among cases (0.44±0.06) as

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Int. J. Life. Sci. Scienti. Res., VOLUME 2, ISSUE 1 compared with controls (0.96±0.04). This finding was similar to

synthase). The Journal of Pharmacology and Experimental

[18, 19]

Therapeutics, 2002; 300:762-7.

Proteins are another potential target of ROS, whose

structure and function can be affected by modification. There are many side chain targets for protein oxidation including cysteine, methionine, and tyrosine. Carbonyls are the oxidation product of proteins and are reported as the potent biomarker of oxidative stress.

[20]

In the present study, Vitamin E level was significantly

(p<0.0001) lower among cases (0.69±0.08) as compared with controls (1.06±0.25). This is in agreement with the study by

[21]

Vitamin E protect against damage to endothelial function from

[4] Carr A, Frei B (Does Vitamin C act as a pro-oxidant under physiological conditions), Federation of American Societies for Experimental Biology, 1999; 13:1007–24. [5] Rathaus A, Bernheim J (Oxygen species in the microvascular environment: regulation of vascular tone and the development of hypertension). Nephrol Dial Transplant, 2002; 17: 216-21. [6] Tandon R, Sinha MK, Garg H, Khanna R, Khanna HD (Oxidative stress in patients with essential hypertension). The National Medical Journal of India, 2005; 18(6):297-9.

elevated blood sugar, particularly in subjects with hypertension.

[7] Kumar KV, Das UN (Are free radicals involved in pathobiology of

The endothelium is the lining of blood vessels, which plays an

human essential hypertension) Free Radic Res Commun, 1993;

important role in blood vessel barrier function, inflammation, blood clotting, and vascular tone and blood pressure. The Centers for Disease Control and Prevention (CDC) report that approximately 67 million American adults, about one in three, have high blood pressure. was

[22]

One of the limitations of this study

smaller sample size, the studied with larger sample size is

19:59-66. [8] Schafer FQ, Buettner GR (Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple).

Free Radical Biology and Medicine.,

2001;

(11):

1191–212. [9] Valko M, Morris H, Cronin MT (Metals, toxicity and oxidative stress). Current Medicinal Chemistry, 2005; 12(10):1161–208.

being recommended for better interpretation of the results.

[10] Satoh, K. (1978). Clinica. Chemica. Acta. 37-43.

CONCLUSION

[11] Navneet Omprakash Soni ‘‘Antioxidant assay in vivo and vitro’’,

This study has shown that the levels of oxidant namely MDA is significantly increased and the levels of enzymatic (SOD) and

International Journal of Phytopharmacology, 5(1), 2014, 51-58. [12] Emmerie A., Engel C (Colorimetric determination of tocopherol (vitamin E)). Rec. Trav. Chim, 1938; 57:1351.

non-enzymatic (Vit E) antioxidants are also markedly decreased

[13] Aquil Ahmad, UshaSinghal, Mohd MobarkHossain, Najmul islam,

in hypertensive patient. The increased concentration of oxidant

Imran Rizvi.The Role of the Endogenous Antioxidant Enzymes

MDA and decreased concentration of antioxidants SOD and

and Malondialdehyde in Essential Hypertension. Journal of Clinical

Vitamin E, supports the hypothesis that lipid peroxidation is an

and Diagnostic Research. 2013;7(6): 987-990

important causative factor in the pathogenesis of hypertension.

[14] Sierra A, Larrousse M (Endothelial dysfunction is associated with

Finally, the alteration in the function of endothelium along with

increased level of biomarkers in essential hypertension). Journal of

antioxidant/pro-oxidant imbalance in hypertension can lead to detrimental consequences and long term adverse effects like atherosclerosis and cardiovascular disease. More extensive study

Human Hypertension, 2010; 24:373-9. [15] Simic DV, Mimikoka J, Pljesa EM, Savic RA, Opacic M, Matic D, et al. (Byproducts of oxidative protein damage and antioxidant enzyme activities in plasma of patients with different degrees of

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essential hypertension). Journal of Human Hypertension, 2006;

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20:149–55.

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